Figure 4.

Transcription of MT-I gene in vitro in the nuclear extract from Rat 1 and R80-1 cells. (A) MT-I transcription in Rat 1 nuclear extract. Nuclear extract (10 μg) from Rat 1 cells was incubated with the template pMT(C₂AT) along with ATP, CTP, 3–0-methyl GTP, and [α-³²P]UTP under optimum conditions at 30°C for 45 min (for details, see ref. 32). The [³²P]UMP-incorporated RNA was isolated, dissolved in RNA loading buffer, and separated by urea-PAGE and analyzed by autoradiography. Lane 1 indicates the level of ³²P-labeled MT-I transcript in Rat 1 nuclear extract, whereas lanes 2 and 3 denote the amount of RNA transcribed when the extract was preincubated with 200 and 400 ng of MRE-s (34 bp) oligo, respectively, and lanes 4 and 5 represent the MT-I transcript level in the presence of 200 and 400 ng of 37 bp rat rDNA enhancer (E₁), respectively, as the competitor. Arrow indicates the transcript. (B) Transcription from MT-I promoter in the nuclear extracts from Rat 1 and R80-1 cells. Identical amounts of the nuclear extract from these cells were analyzed in in vitro transcription reaction as described in A. Lanes 1 and 2 represent MT-I transcript level in 20 and 30 μg of the nuclear extract from Rat 1 cells, whereas lanes 3–5 denote MT-I transcript level in 20, 30, and 40 μg of the nuclear extract from R80-1 cells, respectively.